Agglutination reactions are known to produce aggregates that can be visually or instrumentally detected for qualitative or quantitative assay of biological materials. Agglutination reactions for antigens usually involve antibodies or other binding agents having at least two combining sites specific for multiple, complementary sites on their corresponding antigens or binding partners. These antigens can be found associated with bacterial and mammalian cell surfaces, viral capsids and envelopes, and as soluble and insoluble materials of biological interest such as proteins, carbohydrates, and nucleic acids. Alternatively, agglutination assays for antibodies having a minimum of two antigen-reactive sites are accomplished by adding the multivalent antigen to a solution containing the antibodies. Also useful are agglutination inhibition assays in which a known quantity of labelled, multiepitopic antigen or multivalent antibody competes with an unknown quantity of antigen or antibody, respectively, for combining sites on the binding partner, thereby reducing the extent of agglutination.
The extent of agglutination reactions is known to depend upon the relative concentrations of binding partners. Optimal concentration ranges for each can be empirically determined to provide conditions under which extensive cross-linking of reactants occurs. This cross-linking of individual binding partners provides efficient light scattering aggregates, which can be visually or spectrophotometrically detected. A molar excess of either binding partner relative to the other diminishes or eliminates the cross-linking agglutination reaction in a phenomenon known as the prozone effect, which reduces assay sensitivity. Therefore, maximum assay sensitivity is assured by adjustment of binding partner concentrations into optimal ranges as determined by maximal changes in the light scattering properties of an agglutination or agglutination inhibition reaction.
Sensitivity enhancement has been achieved in agglutination-based immunoassays by attaching immunoreagents to particulates whose improved light scattering efficiency provides additional changes in light scattering signal for each agglutination event. Immunoreagents have been adsorbed onto particulate materials such as latex shperes, or covalently bonded to specific functional groups on particle surfaces for improved stability. U.S. Pat. No. 4,064,080, issued Dec. 20, 1977, discloses styrene polymers with terminal aminophenyl groups having proteins attached to them. U.S. Pat. No. 4,181,636, issued Jan. 1, 1980, discloses carboxylated latex polymers coupled to immunologically active materials through a water soluble activating agent and their use as diagnostic reagents in agglutination tests.
Improved latex reagents are disclosed in U.S. Pat. No. 4,401,765, issued Aug. 30, 1983, comprising shell-core latex polymer particles of 0.03-0.1 .mu.m diameters having a high refractive index polymer core and a polymer shell containing reactive groups such as epoxy, carboxyl, amino, hydroxyl, or formyl groups for covalent coupling of proteins. These improved latexes provide a high refractive index core which maximizes light scattering efficiency while also providing selected functional groups for hapten and protein immobilization onto reactive shells.
Core-shell latex particles of 0.05-1.0 .mu.m diameters having active halogen monomers copolymerized with other ethylenically unsaturated monomers in the particle shell are disclosed in U.S. Pat. No. 4,017,442, issued Apr. 12, 1977. These particles are characterized by having cationic or nonionic surfaces generated by use of surfactants during or after particle polymerizations. Microparticles comprising monodisperse latex beads of 0.5 .mu.m mean diameter and containing copolymerized chloromethylstyrene are commercially available from Polysciences, Inc., Warrington, PA., 18976, for use with covalently bound antigens and antibodies in agglutination tests. These particles are made from styrene copolymerized with chloromethylstyrene and cross-linked with divinylbenzene. U.S. Pat. No. 4,056,501, issued Nov. 1, 1977, discloses further treatment of the particle reagents prepared according to U.S. Pat. No. 4,017,442, previously cited, with nucleophilic groups such as dialkylsulfides or quaternary amines to react with the halogenated latexes to form stable, dispersed particle suspensions in aqueous media. These suspensions were shown to be useful for coatings and organic pigments.
Many of the functional groups found on particle reagents of the prior art are often less than optimal for protein immobilization. For example, some polymeric latex particles having functional groups such as carboxyl and amino groups, require activation prior to protein coupling. The outcome of such a process is variable with respect to the degree of activation and the stability of proteins once they are attached. In other cases such as with epoxy groups, activation is not required, but the active functional groups on the particles are hydrolytically unstable. This results in variably reactive particles that can provide a low concentration of covalently attached proteins. Finally, with functional groups which are either themselves reactive (autoreactive) or require an activation process, the protein coupling conditions may be too stringent requiring the use of elevated temperatures over long periods in the presence of surfactants to effect covalent protein attachment to particles. Such conditions can result in protein denaturations, which can be tolerated only if antigenic identity is sufficiently maintained to allow recognition by complementary binding partners, such as antibodies. However, immobilization of functional proteins such as enzymes, antibodies, specific binding proteins, etc. must be performed in a benign manner in order to preserve their binding or reactive properties.
There is a need for high refractive index particle reagents for use in specific binding assays, especially immunoassays, that provide reactive groups for covalent protein attachment under mild conditions that preserve protein functional or antigenic integrity. This is especially critical when proteins of interest are scarce or have multiple subunits with active binding regions which might be rendered less active by disassociation under severe coupling conditions. Having autoreactive functional groups capable of coupling to proteins under mild conditions and which remain stable and active upon storage in aqueous media and in the presence of other reagents such as detergents and salts, would minimize reagent manipulations and maximize product reproducibility from separate syntheses. A core-shell particle having stable, autoreactive shell functional groups that covalently immobilize proteins under mild coupling conditions, would be highly desireable to provide useful reagents for light scattering specific binding assays.